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Symposium 5: How Proteins Move on DNA

992-SympHigh-resolution Optical Trap Measurements Of A Ringed DNA Translo-caseYann R. Chemla1,2, Jeffrey R. Moffitt2, K. Aathavan2, Shelley Grimes3,Paul J. Jardine3, Dwight L. Anderson3, Carlos Bustamante2,4.1University of Illinois, Urbana-Champaign, IL, USA, 2University ofCalifornia, Berkeley, CA, USA, 3University of Minnesota, Minneapolis, MN,USA, 4Howard Hughes Medical Institute, Berkeley, CA, USA.Ringed ATPases are ubiquitous in the cell, involved in such diverse tasks as celldivision, chromosome segregation, DNA recombination, strand separation, andconjugation. Despite their importance, the mechanism of coordination betweenthe subunits of ringed ATPases remains poorly understood. Viral DNA packag-ing, a process driven by a ringed, homomeric DNA translocase, providesa model system to study this coordination. The Bacillus subtilis bacteriophagephi29 packaging motor has been proposed to operate by sequential action of itssubunits, each packaging DNA in stepwise increments of 2 base pairs (bp) perATP hydrolyzed. Here, we present the first direct observations of individualpackaging steps of this motor with recently developed high-resolution opticaltweezers. Surprisingly, we find that packaging occurs in increments of 10 bp,each consisting of four 2.5-bp substeps. The dwell time statistics between these10-bp ‘‘bursts’’ indicate that the motor loads multiple ATPs prior to transloca-tion, and suggest a two-phase mechanochemical cycle in which ATP bindingand translocation are temporally segregated. These studies reveal a much morecomplex coordination between motor ATPases than previously anticipated thatcontrasts sharply with most published models. In addition, the observation of anon-integer step size demands new models for motor-DNA interactions. Ourfindings are not only relevant to other viral packaging systems, but may alsoprovide insight into the mechanisms of the large class of ringed molecularmotors that share many structural similarities.

993-SympHigh throughput assays for visualizing individual protein-DNA interac-tionsEric Greene.Columbia University, New York, NY, USA.

994-SympCoupling of two motors: T7 helicase-primase and DNA polymeraseSmita Patel1, Manjula Pandey1, Salman Syed2, Taekjip Ha2,Daniel Johnson3, Michelle Wang3.1UMDNJ RWJ Medical School, Piscataway, NJ, USA, 2University of Illinois,Urbana-Champaign, IL, USA, 3Cornell University, Ithaca, NY, USA.T7 bacteriophage encodes all the proteins it needs to replicate its duplex DNAgenome. The T7 replisome consists of the homo-hexameric T7 gp4 helicase-primase protein, T7 DNA polymerase (T7 gp5 complexed with E. coli Thiore-doxin) and the T7 gene 2.5 protein (the single stranded DNA binding protein).The simplicity of the T7 replisome makes it a model enzyme complex to under-stand the mechanisms of DNA replication and the roles of the motor proteins inDNA replication. T7 helicase is a ring-shaped motor protein that binds ssDNAwithin its central channel and uses the energy of dTTP hydrolysis to move proc-essively along ssDNA as well as to unwind the strands of duplex DNA. In thismanner, the helicase motor unwinds the dsDNA genome and creates ssDNAtemplates for the DNA polymerase to copy the duplex DNA strands via leadingand lagging strand synthesis. We will present here our transient-state ensembleand single molecule kinetic data that characterized the movement (speed anddirectionality) and energy coupling (bp translocated per NTP hydrolyzed) ofT7 helicase as a function of duplex DNA stability, the type of nucleotide fuel-ing movement, and how the helicase activity is influenced by the presence ofthe DNA polymerase and primase.

995-SympTranslocation and Unwinding by DnaBOmar A. Saleh.University of California, Santa Barbara, Santa Barbara, CA, USA.DNA replication requires the action of a helicase, a motor protein that unwindsdsDNA into its single-stranded components. In E. coli, the replicative helicaseis DnaB, a hexameric, ring-shaped protein that encircles and translocates alongssDNA, denaturing dsDNA in advance of its motion. Using multiplexed single-molecule measurements with a magnetic tweezer (Ribeck and Saleh, 2008), wehave investigated the translocation and unwinding activities of DnaB, bothalone and in complex with other replisome components. We discuss our find-

ings in the context of both ‘Brownian ratchet’ theories of dsDNA denaturationand mechano-chemical theories of motor protein motion.

Symposium 6: Store-operated Calcium Channelsin the Molecular Age

996-SympSTIMulating Calcium Entry at ER-Plasma Membrane JunctionsJen Liou, Onn Brandman, Tobias Meyer.Stanford University, Stanford, CA, USA.Store-operated calcium (SOC) entry is essential for maintaining endoplasmicreticulum (ER) functions and generating the sustained calcium signals crucialfor gene expression, secretion, cell motility, and cell proliferation. To under-stand the molecular mechanism of how receptor-evoked ER calcium store de-pletion induces calcium entry through SOC channels at the plasma membrane,we screened a small interference RNA (siRNA) library targeting the humansignaling proteome and identified several regulators of SOC entry includingSTIM1 and STIM2. We found that STIM1 initiates SOC signaling by sensingcalcium levels in the ER using its luminal EF-hand motif. Using a fluorescenceresonance energy transfer (FRET) approach, we showed that STIM1 rapidlyforms oligomers after calcium store depletion. We further demonstrated thatSTIM1 subsequently translocates to ER-plasma membrane junctions usingconfocal and total internal reflection fluorescence (TIRF) microscopy. More-over, we showed that STIM1 translocation to ER-plasma membrane junctionsrequires a C-terminal polybasic motif. We further conducted human siRNAscreens for regulators of basal calcium homeostasis and identified STIM2 asa feedback regulator that stabilizes basal cytosolic calcium levels. We showedthat STIM2, like STIM1, senses ER calcium levels by its EF-hand motif,translocates to ER-plasma membrane junctions, and activates Orai1 calciumchannels. In contrast to STIM1, STIM2 is activated in response to smaller re-ductions in ER calcium levels. Our data support two conclusions: 1) STIM2functions independently of STIM1 for basal ER calcium levels or weak recep-tor stimuli and 2) STIM2 acts synergistically with STIM1 when calciumstores are depleted by strong receptor stimuli. We are further investigatingthe plasma membrane targeting mechanism of STIM1/STIM2 and are charac-terizing other regulators of SOC entry that we have identified in the siRNAscreens.

997-SympCalcium Signals In Lymphocyte Activation And DiseaseStefan Feske.NYU Medical Center, New York, NY, USA.Calcium ions function as universal second messengers in virtually all eukary-otic cells including cells of the immune system where they are crucial for thefunction of T and B cells, mast cells and dendritic cells. The predominantmechanism regulating intracellular Ca2þ levels in cells of the adaptive im-mune system is store-operated Ca2þ influx through so-called Ca2þ�releaseactivated Ca2þ (CRAC) channels. We identified ORAI1 (also namedCRACM1) as a pore subunit of the CRAC channel essential for the functionof T cells and mast cells. Mutation of ORAI1 in humans is associated withsevere combined immunodeficiency (SCID), increased susceptibility to infec-tions and a failure to thrive. ORAI1/CRAC channels are activated when intra-cellular Ca2þ stores are depleted. The resulting decrease in the ER Ca2þconcentration is sensed by stromal interaction molecule 1 (STIM1) that is re-quired for activation of ORAI1/CRAC channels. We showed that murineT cells lacking STIM1 exhibit severely impaired store-operated Ca2þ influx.T cells from mice lacking STIM1 or its paralogue STIM2 both showed signif-icantly reduced cytokine production in vitro and a defect in regulatory T celldevelopment as well as lympho- and myeloproliferation in vivo. T cells frommice transgenic for an R91W mutation in ORAI1 that abrogates CRAC channelfunction in T cells from human SCID patients also showed substantially im-paired store-operated Ca2þ influx and cytokine gene expression. This impor-tant role of STIM1 and ORAI1 in T cell function in mice is also apparent inT cells from human patients with SCID, which lack expression of ORAI1and STIM1, respectively. Taken together STIM1, STIM2 and ORAI1 are es-sential regulators of store-operated Ca2þ entry in cells of the immune systemand other tissues.

998-SympStructural And Mechanistic Insights Into Stim1-mediated Initiation OfStore Operated Calcium Entry.Mitsu Ikura.

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Division of Signaling Biology, Ontario Cancer Institute and Department ofMedical Biophysics, University of Toronto, Toronto, ON, Canada.Stromal interaction molecule-1 (STIM1) activates store operated Ca2þ entry(SOCE) in response to diminished luminal Ca2þ levels. We have recently de-termined the solution structure of the Ca2þ�sensing region of STIM1 consist-ing of the EF-hand and sterile a motif (SAM) domains (EF-SAM) (Stathopuloset al. Cell Oct 3rd issue, 2008). The canonical EF-hand is paired with a previ-ously unidentified EF-hand. Together, the EF-hand pair mediates mutually in-dispensable hydrophobic interactions between the EF-hand and SAM domains.Structurally critical mutations in the canonical EF-hand, ‘hidden’ EF-hand orSAM domain disrupt Ca2þ sensitivity in oligomerization via destabilizationof the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mu-tations within full-length STIM1 induce punctae formation and activate SOCEindependent of luminal Ca2þ. We provide atomic resolution insight into themolecular basis for STIM1-mediated SOCE initiation and show that thefolded/unfolded state of the Ca2þ sensing region of STIM is crucial toSOCE regulation. (Supported by CIHR and CFI).

999-SympA Molecular Mechanism for CRAC Channel ActivationRichard S. Lewis.Stanford University, Stanford, CA, USA.The Ca2þ release-activated Ca2þ (CRAC) channel is the most intensively stud-ied member of the class of store-operated channels, and is well known to playa critical role in lymphocyte activation during the immune response. The cen-tral mystery of store-operated Ca2þ entry (SOCE) has been how depletion ofCa2þ within the ER lumen triggers CRAC channel activation in the plasmamembrane (PM). Recent breakthroughs in identifying the ER Ca2þ sensor,STIM1, and the pore-forming subunit of the CRAC channel, Orai1/CRACM1,have spurred rapid progress in defining a molecular mechanism. Store depletiontriggers the oligomerization of STIM1 and its redistribution to ER-plasmamembrane (ER-PM) junctions, where Orai1 accumulates in the plasma mem-brane and CRAC channels open. These dynamic structures, consisting of clus-ters of STIM1 and Orai1 separated by a 10-25 nm gap comprise the elementaryunits of SOCE. Using fusion proteins in which the Ca2þ sensing domains ofSTIM1 were replaced by FRB and FKBP12, we showed that rapamycin-in-duced oligomerization causes the proteins to accumulate at ER-PM junctionsand activate CRAC channels without Ca2þ store depletion. Thus, STIM1 olig-omerization itself acts as a master switch to trigger the self-organization andactivation of the SOCE machinery. Oligomerization acts by enabling STIM1to accumulate at ER-PM junctions and to bind to CRAC channels diffusingthroughout the overlying PM. Orai1 is trapped and activated by binding to a cy-tosolic subregion of STIM1 that we call the CRAC activation domain, or CAD.In vitro studies with purified proteins show that CAD binds directly to purifiedOrai1 and crosslinks CRAC channels to form clusters. These studies supporta molecular mechanism for SOCE by which the STIM1 CAD traps and acti-vates CRAC channels at ER-PM junctions via direct binding to Orai1.

Platform L: Membrane Protein Structure

1000-PlatHigh Throughput Coarse-Grained Simulations of the Insertion of Trans-membrane HelicesBenjamin A. Hall, Alan Chetwynd, Richard Franzese, Mark S.P. Sansom.University of Oxford, Oxford, United Kingdom.Transmembrane helices play multiple vital roles in cell function, including sig-nalling processes, channel gating and active transport. As such, there existsa significant body of data on the biological function and structural propertiesof naturally occurring helices and their mutants, and on synthetic helicessuch as WALP and LS3 which have been used to better understand the rolesof different residues in determining position and orientation of helices ina membrane. Coarse grained MD simulations are becoming an increasinglypopular tool for understanding the properties of biological systems, overcomingcanonical limits of atomistic simulations such as timescale or system size. Suchtechniques involve several manual steps, including system build, simulation setup and analysis. Here we present the Sidekick software, which enables automa-tion of these processes, thus enabling high throughput simulations on the basisof a small set of input sequences, or of a single sequence and a scanning mu-tation. We demonstrate the use of this software to approach two problems;the ability of two commonly used coarse grain methods to predict insertion ef-ficiencies of helices generated from sliding window across a larger sequence,and molecular signalling of TM2 of the methyl accepting chemoreceptor pro-tein Tar. Our results demonstrate the value of such an HT simulation approachin the interpretation of a range of experimental data.

1001-PlatStructural models of Alzheimer’s Abeta channelsH.R. Guy, Stewart R. Durell, Yinon Shafrir.NCI, NIH, Bethesda, MD, USA.Amyloid beta (Ab) peptides involved in Alzheimer’s disease form Ca2þ perme-ant ion channels. We have been developing models of these channels to be con-sistent with experimental findings. These models are refined and evaluated bymolecular dynamics (MD) simulations. In our segment nomenclature, residues1-14 are called S1, residues 15-28 S2, and residues 29-42 S3. The models wefind most consistent with functional properties have a pore lining formed by 6-12 S1 segments in a b-barrel structure. The cation selectivity of the pore isdue to negatively charged residues at positions 1,3,7, and 11 that extend intothe pore. This category of models may differ by the number of strands, whetherthese strands are parallel or antiparallel, and the structures of the protein that sur-round the S1 b-barrels. Heavy metal ions and histidine-containing peptides blockAb channels and inhibit Ab-induced apoptosis. Zn2þ and Cu2þ binding sites areformed in our models by E11, H13, and H14 residues. Clustering of these residuesat the entrance to the pore is more pronounced in the parallel models, and they arenearer the axis of the pore in the 6-stranded models. The 6-stranded b-barrelmodels are also more consistent with blockade of the channels by Tris cations.We have developed hexameric and dodecameric models in which S2 and S3 seg-ments are helical, and dodecameric models in which S2 and S3 segments forma 24-stranded b-barrel that surrounds the pore-forming S3 b-barrel. However,the models most consistent with microscopy studies of the channels are composedof 36 subunits with only a fraction of the S1 segments forming the pore. Thesemodels are hexamers of hexamer in which S3 segments of each hexamer formsa 6-stranded b-barrel that is exceptionally stable during MD simulations.

1002-PlatPore Formation and Structure of the Twin Arginine Translocase SubunitTatA from B. subtilisStephan L. Grage1, Torsten H. Walther1, Claudia Muhle1, Olga Nolandt1,Marco J. Klein1, Nadine Roth1, Sonja D. Mueller1, Philip Callow2,Anna de Angelis3, Fabian V. Filipp3, Stanley J. Opella3, Anne S. Ulrich1.1Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Institut LaueLangevin, Grenoble, France, 3University of California, San Diego,San Diego, CA, USA.Cells have developed sophisticated transport machineries to allow proteins tocross membrane barriers. In bacteria, the twin arginine translocase (Tat) cantranslocate proteins even in the folded state. In B. subtilis, a membrane proteincomplex consisting of two subunits, TatA and TatC, is responsible for the Tattranslocation process. TatA is believed to form a nanometer size pore troughwhich the protein is transported, whereas TatC is involved in recognition ofthe target protein signal peptide.To get insight into the mechanism of the Tat translocation in B. subtilis, we stud-ied the structure of the pore-forming subunit TatA and the pore assembly pursu-

ing two complementary experimentalapproaches. The structure of individualTatA monomers in membranes or mem-brane-mimetic environments was char-acterized using solid state and solutionNMR. The formation of oligomeric as-semblies of TatA in the membrane, onthe other hand, was investigated usingin-plane neutron scattering of TatA re-constituted in aligned membranes. Thisway, we were able to both derive a de-tailed structural model of TatA, and tocharacterize pores formed by TatA.

1003-PlatLysophospholipid Micelles Sustain Diacylglycerol Kinase in Active andStable Form for Biochemical and Structural StudiesEndah S. Sulistijo, Charles Ellis, Megan Wadington, Charles R. Sanders.Vanderbilt University, Nashville, TN, USA.One of the challenges in the study of membrane proteins is that many methodsrequire the proteins be soluble and stable in aqueous solution, which can besolved by the use of detergent micelles to provide an environment that mimicsthe hydrophobic environment of biological membranes. Yet, it remains to bedetermined which detergents are the best in sustaining the structural conforma-tion and biological function of the proteins of interest. In this study, we usedEscherichia coli DAGK, which is an integral membrane protein, to screen de-tergent conditions that can maintain the catalytic activity of the protein and aresuitable for structural studies using NMR spectroscopy. We learned that